Neural precursor-specific expression of multiple Drosophila genes is driven by dual enhancer modules with overlapping function

Abstract

Transcriptional cis-regulatory modules (CRMs), or enhancers, are responsible for directing gene expression in specific territories and cell types during development. In some instances, the same gene may be served by two or more enhancers with similar specificities. Here we show that the utilization of dual, or "shadow", enhancers is a common feature of genes that are active specifically in neural precursor (NP) cells in Drosophila. By genome-wide computational discovery of statistically significant clusters of binding motifs for both proneural activator (P) proteins and basic helix-loop-helix (bHLH) repressor (R) factors (a "P+R" regulatory code), we have identified NP-specific enhancer modules associated with multiple genes expressed in this cell type. These CRMs are distinct from those previously identified for the corresponding gene, establishing the existence of a dual-enhancer arrangement in which both modules reside close to the gene they serve. Using wild-type and mutant reporter gene constructs in vivo, we show that P sites in these modules mediate activation by proneural factors in "proneural cluster" territories, whereas R sites mediate repression by bHLH repressors, which serves to restrict expression specifically to NP cells. To our knowledge, our results identify the first direct targets of these bHLH repressors. Finally, using genomic rescue constructs for neuralized (neur), we demonstrate that each of the gene's two NP-specific enhancers is sufficient to rescue neur function in the lateral inhibition process by which adult sensory organ precursor (SOP) cells are specified, but that deletion of both enhancers results in failure of this event.neural precursors | dual enhancer modules | cis-regulatory code | proneural proteins | bHLH repressors S pecification of neural precursor (NP) cell fates is a core step in the process of neural development, and there has long been intense interest in its mechanistic basis. Among the most heavily investigated questions in this arena is how NP-specific expression of key regulatory factors associated with the NP fate is achieved. For example, multiple prior studies have used computational methods to identify NP-specific cis-regulatory modules (CRMs) genome-wide, in an attempt to define common transcription factor inputs that might underlie NP-specific gene expression (1-3).Proneural transcriptional activators of the basic helix-loophelix (bHLH) family are the top-level regulators of NP specification. They confer on cells in ectodermal tissue the potential to adopt the NP fate; loss of proneural gene function results in loss of all NPs and thus loss of expression of all NP-specific regulatory factors. However, proneural factors are not expressed only in NPs; rather, they are initially expressed in small groups of cells called proneural clusters (PNCs). At this stage of the process, most or all cells in the cluster have the potential to become an NP. This is prevented by "lateral inhibition", in which the single NP that will ultimately arise from the PNC inhibits al...

Regulatory logic driving stable levels of defective proventriculus expression during terminal photoreceptor specification in flies

“…Although some enhancers account for distinct aspects of regulation, others drive overlapping patterns. Shadow enhancers can compensate for removal of a primary enhancer, resulting in mostly unaltered gene expression Miller et al, 2014;Nolte et al, 2013;Perry et al, 2012). These shadow enhancers provide reliability and robustness in pattern formation, allowing crucial patterning genes to be buffered against environmental and genetic variation (Barolo, 2012;Bothma et al, 2015;Frankel et al, 2010;Perry et al, 2010).…”

“…Interestingly, we also observed derepression of yR7 enh in outer PRs in dve mutants, suggesting that yR7 enh serves as an inducible backup or 'dark' shadow enhancer in these cells. Shadow enhancers are DNA elements that drive redundant expression patterns and ensure robust gene expression in cases of genetic and environmental perturbation (Bothma et al, 2015;Frankel et al, 2010;Hong et al, 2008;Miller et al, 2014;Nolte et al, 2013;Perry et al, 2010;Wunderlich et al, 2015). yR7 enh represents an unusual 'dark' shadow enhancer as it is normally repressed and only becomes active when Dve driven by the primary outer enh is compromised.…”

Hairless as a novel component of the Notch signaling pathway

“…(Tribolium et al 2008). Senseless directly recruits the CtBP co-repressor via the PLNLS motif (Miller et al 2014). This portion of the protein is encoded in exon 2; splice junction is indicated by a red /.…”

DrosophilaNeuroblast Selection Gated by Notch, Snail, SoxB and EMT Gene Interplay

“…The TFs encoded by the E(spl)-C in turn repress the bHLH proneural genes within the achaete-scute complex (AS-C) i.e., achaete (ac), scute (sc) and lethal of scute (l'sc) (Giagtzoglou et al, 2003). In addition, the E(spl)-C also represses the neur and Dl genes, as well as the asense (ase) gene; a proneural-related bHLH gene located within the AS-C (Heitzler et al, 1996;Miller and Posakony, 2018;Miller et al, 2014). Downregulation of the proneural genes inhibits NB formation, instead promoting epidermal differentiation (Jimenez and Campos-Ortega, 1990).…”